direct inoculum protocol (dip) for bacterial identification using an automated microbial identification and antimicrobial susceptibility testing system (amiast)

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Sepsis, an important global health problem, is defined as life-threatening organ dysfunction due to a dysregulated host response to infection. The Global Burden of Diseases Study estimated that there were 48.9 million cases of sepsis and 11 million sepsis-related deaths worldwide in 2017, which accounted for almost 20% of all global deaths1. Around 2/3rd of bloodstream infections (BSI) causing mortality are due to gram-negative bacterial pathogens2. The leading causes of mortality amongst gram-negatives (GN) are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii,&#160;which account for around 40% of cases amongst 33 bacterial pathogens2.
Blood cultures remain the gold standard for diagnosing BSI, and rapid microbial identification along with antimicrobial susceptibility testing (AST) results is the key to management. It has been estimated that there is a 9% increase in odds of mortality with each-hour delay in instituting appropriate antimicrobials in sepsis3. The turnaround time (TAT) of microbiologically positive blood culture reports with AST results is around 48-72 h with the available microbiological tools in resource-limited settings, even with automated systems. As a result of this subpar TAT, broad-spectrum antimicrobials are used empirically, contributing to the burgeoning problem of antimicrobial resistance (AMR). Recognizing this dire need to reduce TAT for microbiological culture techniques for sepsis, EUCAST and CLSI are moving towards performing AST directly from positively flagged blood culture bottles (+aBC)4,5.
In 2018, EUCAST first introduced the rapid AST (RAST) method for determining AST by Kirby-Bauer disk diffusion method at short incubation times, i.e., 4 h, 6 h and 8 h, directly from +aBC6,7. The method is presently validated for determining AST for +aBCs containing one of the 8 most common causes of BSI namely E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii complex&#160;amongst gram-negatives and Staphylococcus aureus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae amongst gram-positives8.The breakpoints for AST determination at various time intervals are provided as per microbial species listed above. Hence, before categorical interpretation of AST results, microbial identification is necessary. However, the RAST standard does not specify the method to enable microbial identification within this time frame.
The majority of studies evaluating the EUCAST RAST method in their setting have used mass spectrometry-based microbial identification after short incubation on plated media to identify micro-organisms9,10,11,12,13,14,15,16,17. However, mass spectrometry instruments are not widely available, especially in low to middle-income countries (LMICs), which greatly limits the potential usefulness of this method. Few studies have reported implementation of this method in their centers without using mass spectrometry18,19,20. Tay&#351;i et al.18 reported a broad categorization of GN amongst Enterobacterales, Pseudomonas, and Acinetobacter spp. based on gram stain morphology and oxidase test before interpreting AST results. In other studies from this center, by Gupta et al.19 and Siddiqui et al.20, species-level microbial identification was done by preparing a bacterial pellet from the positively flagged blood-broth mixture and inoculating it on the conventional biochemical tests. While Tay&#351;i et al.18 did not comment upon the accuracy of microbial identification with their approach, Gupta et al.19 reported that with their approach in 165/176 (94%) cases, a RAST reportable gram-negative (RR-GN), i.e., either of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii complex. However, with the latter approach, the reading of RAST results was done retrospectively using 8 h zone diameter breakpoints only after the full incubation of conventional biochemical results i.e., 18-24 h post-inoculation, and the average time for reporting was around 2 days.
To reduce the TAT of clinical reports further, we propose an alternative methodology to enable early identification of GN present in +aBCs using aMIAST. Before the introduction of mass spectrometry-based microbial identification systems, these automated identification systems were considered the standard of care for microbial identification, where identification was enabled by colorimetric and/or fluorometric changes induced by test bacteria when inoculated in miniaturized biochemical tests harbored in a cassette and matching the results with their isolate database. The average time to identification in these systems is around 4 h to 8 h however, they are limited by the fact that the manufacturers recommend overnight growth of microbes before their respective identification cards can be inoculated. This requirement greatly limits their usefulness in reducing the time to report.
Few studies have evaluated methods to directly identify microbes from +aBCs using these automated systems21,22,23,24,25,26,27. In the case of +aBCs containing monomorphic GN, the majority of studies showed excellent concordance between direct inoculation from bacterial pellet made from positive blood-broth mixture and standard colony incubation. However, in the case of gram-positives, the concordance rates were suboptimal. As the average time to positivity of +aBCs is between 8 h and 16 h and identification of GN takes ~4 h to 8 h in an automated microbial identification system, we hypothesize that by employing direct inoculation protocol in the automated microbial identification, we can complete the clinical reporting of +aBCs with GN having a RR-GN within 24 h of sample receiving.
Setting for the study 
The present study was conducted in the clinical bacteriology laboratory of a 950-bed, academic, tertiary care institute of national importance (INI) in Central India from January to October 2023. The laboratory is equipped with a continuous blood culture monitoring system (CBCMS) and aMIAST. The bacteriology laboratory is functional round-the-clock with the availability of technicians and microbiologists for processing and reporting any positively flagged blood culture bottle (+aBCs).
Microbial methods used here 
The workflow of the study is shown in Figure 1. The +aBCs showing monomorphic GNs (+naBC) were processed by direct inoculation of corresponding identification cards to enable identification and AST using EUCAST RAST protocol. These results were compared with the standard-of-care (SoC) method for +aBCs i.e., subculturing on conventional plated media through sheep blood agar (SBA), chocolate agar (CA), and MacConkey agar (MA), incubated aerobically for 16 h to 24 h followed by identification and AST cards given by aMIAST when isolated colonies appear. Blood cultures showing gram-positive cocci, gram-positive bacilli, budding yeast cells, and &#8805;2 different micro-organisms on initial gram staining or plated media were excluded from the study.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

This research aims to evaluate the diagnostic accuracy of Gram-negatives directly from positively flag blood culture bottles by automated microbial identification system. The purpose is to utilize it as a tool for early clinical reporting by EUCAST rapid antimicrobial susceptibility testing method. EUCAST RAST method was recently introduced in 2018 to enable reporting of anti-microbial susceptibility testing results within four to eight hours of positive flagging of automated blood culture bottles.It involves performing everywhere disk diffusion method directly from positively flagged bottle and reading results at either four, six, or eight hours. For clinical reporting by EUCAST RAST method, the microbial identification is a prerequisite for determining the interpretative category of AST results. The major challenge in implementing this method is early identification of the microbes within eight hours, especially in resource poor settings, which lacks mass spectrometry.We found that the direct inoculum protocol was around 94%accurate in identifying a RAST reportable Gram-negative, which includes Escherichia coli, Kiebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex. This approach of identifying Gram-negatives directly was around four times faster than the standard approach of identification after overnight incubation. We will now focus on assessing the impact of this approach of clinical reporting in Gram-negative sepsis on patient outcomes in terms of length of hospitalization and mortality rate.Furthermore, it can also be used as a tool to reduce anti-microbial usage to facilitate anti-microbial stewardship.

Direct Inoculum Protocol (DIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

To begin, swab the septum of an automated blood culture bottle, having monomorphic gram-negative organisms with 70%isopropyl alcohol inside a class 2A biosafety cabinet. Draw five milliliters of blood broth mixture into a sterile syringe equipped with a 21 gauge needle. Next, disinfect the rubber septum of a rubber separator tube.Transfer the blood broth mixture into the tube. Centrifuge the sample at 160 G for 10 minutes. Now, carefully pipette out the supernatant inside a biosafety cabinet.Transfer the supernatant to a new plain blood collection vial. Place the capped vial in a centrifuge at 2000 G for 10 minutes. Now, aspirate the supernatant with a sterile pipette.Then dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle. Using a sterile inoculation loop, pick the bacterial pellet from the bottom of the vial and inoculate it into the aMIAST tube. Use sterile saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63.Then place a capillary attachment of the gram-negative aMIAST identification card in the first tube. Position the selected cards appropriately on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.Close the door and press fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The results of seven positive blood culture bottles, identified using the direct inoculum protocol, were discordant until the species level.Of the 105 RAST reportable gram negatives identified by the standard inoculum protocol, 98 were correctly identified by the direct inoculum protocol. Among 99 non-RAST reportable gram negatives, a RAST reportable gram negative was identified in four positive blood culture bottles.

direct-inoculum-protocol-dip-for-bacterial-identification-using-an

Research

JoVE Journal

Medicine

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Standard Inoculum Protocol (SIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

To begin, swab the septum of an automated blood culture bottle containing monomorphic gram-negative organisms, with 70%isopropyl alcohol, inside a Class II A biosafety cabinet. Then, draw one milliliter of blood broth mixture into a sterile syringe equipped with a 21-gauge needle. Dispense a big drop of the mixture onto the surface of a plated media.After streaking the plates, incubate the cultures at 37 degrees Celsius for 18 to 24 hours. Examine the plates for isolated colonies inside the biosafety cabinet. Next, dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle.Now pick up three to five morphologically similar colonies with a sterile straight inoculation wire. Transfer the inoculum into the first tube. Use saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63 McFarland.Then, place a capillary attachment to the gram-negative aMIAST identification card in the first tube. Position the selected cards on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.Close the door and press Fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The standard inoculum protocol identified 105 out of 204 tested isolates as RAST reportable gram-negatives, with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex being the primary organisms identified.

Antimicrobial Susceptibility Test (AST) Using the EUCAST Rapid Antimicrobial Sensitivity Testing (RAST) 

To begin, place two uninoculated 90 millimeter Mueller-Hinton agar plates in a biosafety cabinet. With gloves on, clean the septum of an automated blood culture bottle containing monomorphic gram-negative organisms with 70%isopropyl alcohol. Use a sterile syringe to aspirate 100 to 150 microliters of undiluted blood broth mixture from the blood culture bottle.Add the sample to the center of a Mueller-Hinton agar plate. Now, use a sterile cotton swab to gently spread the broth over the plates in three directions. Then, apply about six or fewer antimicrobial discs on each plate.Transfer the plates to an aerobic incubator at 35 degrees Celsius for eight hours. When inoculation is complete, observe the purity of the isolate. Read the inhibition zones within eight hours, give or take five minutes.Use the RAST breakpoint table for short incubation to interpret the results after checking the bacterial identification results in the AMI-AST.

Quality Control Testing for Rapid Antimicrobial Sensitivity Testing (RAST)

For the weekly quality control testing of the RAST method, place four sterile glass tubes on a tube stand. Dispense sterile saline into the tubes. Next, transfer the QC strain from the isolated colonies of an overnight culture into the first tube to create a 0.5 McFarland suspension.With a sterile pipette and tip, transfer 10 microliters of the suspension from the first tube to the second tube. Once mixed, transfer 10 microliters of the suspension from the second to the third tube, and then to the last tube. Next, take one milliliter of the inoculum from the last tube with a sterile syringe.Transfer the inoculum into an automated blood culture bottle after swabbing the septum with 70%isopropyl alcohol. Simultaneously with a separate sterile needle, add five milliliters of sterile sheep blood into the same blood culture bottle. Incubate it in a continuous blood culture monitoring system until it flags positive.